BUONGIORNO A TUTTI!!!!!!!!!!
AREVIR-GenaFor-Meeting Verbundprojekt HIV-HEP-MASTER EuResist (8-9/5/2015) Comparison of HIV-1 genotypically predicted tropism between circulating plasma RNA and proviral DNA in drug-experienced patients Lavinia Fabeni National Institute for Infectious Diseases L. Spallanzani - IRCCS, Rome, Italy University of Rome Tor Vergata, Italy
Background • Maraviroc ( MVC ) is the first R5 antagonist approved for the treatment of patients already experiencing virologic failure because of resistance to other antiretroviral agents (Rodger D. et al ., CID, 2008; Sayana S. et al., Expert Rev Anti Infect Ther, 2009). • In the contest of virologic suppression , regimen switching to MVC is now feasible . • In the clinical practice the genotypic tropism testing ( GTT ) is performed using plasma HIV-1 RNA . But in limited settings (as very low or undetectable viral load or in case of lack of plasma) proviral DNA ( pvDNA ) may be considered as a potential alternative source of viral genetic material for tropism testing (Vandekerckhove L. et al ., Lancet Infect Dis, 2011), and would facilitate the use of CCR5 inhibitors as part of switching , simplification , or intensification strategies . • Several studies show a high concordance between HIV-1 genotypic coreceptor tropism prediction based on plasma RNA and pvDNA (Saracino A. et al ., Virus Res, 2007; Seclén E. et al ., JAC, 2010; Verhofstede C. et al ., HIV Med, 2011; Paar C. et al ., J Clin Microbiol, 2011; Gupta S. et al ., JAC, 2013; Bon I. et al ., J Med Virol, 2014), but the factors that could be associated with the discrepancy between the two compartments has not yet been clearly determined.
Objective The aim of this study was to analyse the degree of GTT concordance between plasma HIV-RNA and pvDNA and to assess which factors may affect the discrepancy between the two compartments .
Materials & Methods
Extraction, , Amplification and Sequencing Paired PBMCs DNA and plasma RNA samples were obtained from 55 drug experienced subjects and were tested for the V3 region of gp120 (during 2010-2014) RNA extraction from plasma by DNA extraction from PBMCs by QIAmp Viral RNA mini Kit QIAmp DNA mini Kit Amplification by home-made RT-PCR Amplification by home-made PCR Amplification by NESTED-PCR Sequencing by Genetic Analyzer 3130 Viral RNA and proviral DNA sequences were compared at the amino acid level
• Patients selected had paired samples with blood sampling dates contextual or within a period of 24 months maximum , according to the observation that no evolution is found in samples within 24 months of follow-up, both for the mutated positions and the total number of mutations (Palmisano L. et al ., HIV Med, 2009). • A phylogenetic analysis on HIV-1 V3 sequences was performed for the following purposes: i) determine viral subtype ; ii) check any cross-contamination ; iii) evaluate the proper clustering of the pvDNA and plasma RNA sequences from the same subject. The robustness of the paired samples clusters was tested using the maximum likelihood (ML) method. This was inferred with the GTR nucleotide substitution model with gamma-distribution of among site rate heterogeneity, a proportion of invariable sites (GTR+I+Г 5) (Tavaré S., Lect Math. Life Sci, 1986), and 1000 bootstrap replicates. Paired samples clusters were identified by a bootstrap support >70% . Genetic distances analyses were conducted using the Tajima-Nei model (Tajima F. and Nei M., Mol Biol Evol, 1984). • Tropism was predicted by Geno2Pheno setting the false positive rate (FPR) at 10% (http://coreceptor.bioinf.mpi-inf.mpg.de/). Differences between the two compartments were evaluated by analysing co-receptor • usage , genetic distance and V3 mutations prevalence . • Paired samples were also stratified in three viremia levels (<50, 51-500 and >500 copies/mL).
Results
Patient’s characteristics at the time of pvDNA V3 genotyping 37/55 couples of samples were contextually available , while 3 C, 3 BF, 2 F1, 2 G, 18/55 had paired samples with 1 BC and 1 CRF06_cpx blood sampling dates within a median [interquartile period, IQR] period of 7 [2-9] months .
By phylogenetic analysis , sequences belonging to the same subject showed a high homology in 100% of cases , denoting genetic relatedness . Plasma RNA pvDNA Maximum likelihood (ML) tree of the 55 RNA/DNA paired samples (bootstrap value >70%)
The concordance between GTT in plasma RNA and pvDNA compartments was found in 46/55 (84%) samples (A). The concordance was very high among contextual samples ( 92.0%, 34/37 ) (B), and still considerable among non contextual samples ( 66.7%, 12/18 ) (C). Discordant samples were characterized by a higher FPR difference between pvDNA and RNA than concordant samples (median [IQR] ΔFPR (%): 20 [11-52] vs. 1 [0-7.6], p<0.001 ) and a higher genetic variability (median [IQR] genetic distance: 0.07 [0.02-0.23] vs. 0.00 [0.00-0.02] p=0.001 ). In general, lower viremia levels were found in discordant samples compared to concordant samples (median [IQR] log 10 copies/mL: 1.73 [1.60-2.02] vs. 2.26 [1.92-4.12], p=0.036 ). 100 A B C 90 80 70 % Samples 60 Concordant 50 92 66.7 83.6 Discordant 40 30 20 10 33.3 16.4 8 0 Overall Contextual Non contextual N (%) 46 (83.6) 9 (16.4) 34 (92.0) 3 (8.0) 12 (66.6) 6 (33.4) p=0.036 Plasma HIV RNA, median (IQR) log 10 copies/mL 2.26 (1.92-4.12) 1.73 (1.60-2.02) 2.85 (1.93-4.23) 1.73 (1.66-3.01) 2.13 (1.45-2.31) 1.62 (0.40-1.92) p<0.001 ΔFPR (%), median (IQR) 1.0 (0.0-7.6) 20.0 (11.0-52.0) 1.1 (0-7) 20.2 (15.8-29.6) 0.8 (0.0-38) 21.0 (8.6-72.0) p=0.001 0.00 (0.00-0.02) 0.07 (0.02-0.23) 0.00 (0.00-0.01) 0.07 (0.05-0.14) 0.02 (0.00-0.03) 0.07 (0.01-0.26) Genetic distance, median (IQR) 0.01 (0.00-0.02) 0 (0-0.02) 0.02 (0.01-0.07) Characterization of HIV-1 tropism, viremia and genetic diversity between RNA/DNA paired samples.
Regarding the: • Number of mutated positions a substantial similarity between plasma RNA and pvDNA was found: Median [IQR]: 7 [5-9] and 6 [5-8], respectively; p=0.82- by Mann-Whitney test • Number of quasispecies increased in pvDNA: Median [IQR]: 0 [0-4] in RNA sequences; 4 [0-8] in DNA sequences; p=0.008 by Mann-Whitney test The archived viruses may not always correspond to the most prevalent virus in plasma
Comparison of the overall G2P FPRs in both plasma RNA and pvDNA compartments showed good correlation (r=0.72, p<0.001) (A). A very good correlation was found for the 37 contextual samples ( r=0.93, p<0.001 ) (B). Among the 18 non contextual samples the correlation was low ( r=0.37 p=0.046 ) (C), explained by a high difference in FPR (%) between the 2 compartments ( Median [IQR]:7.3 [0.2-43.5] ). Scatterplot for the comparison between RNA ( x axis) and DNA ( y axis) V3-based coreceptor tropism prediction.
R5 tropism was predicted in 38/46 The proportion of X4/DM tropic virus (82.6%) couples of samples, with an FPR in discordant samples was significantly ranging from 13.0% to 96.1% and higher in pvDNA than in plasma RNA X4/DM in 8/46 (17.4%), with an FPR ( 8/9 [88.9%] vs. 1/9 [11%], P=0.0034 ). ranging from 0.2% to 8.6%. Overall tropism of concordant samples (A); X4/DM tropism of discordant samples (B).
The prevalence of discordant samples increased with the decrease of viremia levels Evaluation of tropism concordance between RNA/DNA paired samples according to different viremia levels among the 42 paired samples with the same viremia level. p=0.2, by Squared-test for trend - any statistical significance was shown, probably due to the low number of samples analysed per each group
The prevalence of X4/DM samples increased with the decrease of viremia levels Evaluation of X4/DM tropism among the 36 couples of samples with concordant tropism and the same viremia level. p=0.052 by Squared-test for trend.
No difference between the two compartments in the mutation prevalence was found in R5 concordant samples. Prevalence of V3 mutations in R5 concordant DNA/RNA samples (N=38). All mutations found at the 35 V3 positions with an overall prevalence ≥ 10% were evaluated.
In X4/DM concordant samples , the V3 mutations ( S11G, H13R, E25R, Q32K, H34Y ) were found with a higher prevalence in DNA samples , while the V3 mutations ( N5D, K10R, H13S, T22A, G24R, E25K, I26V ) were mostly prevalent in plasma RNA than pvDNA, suggesting a different viral evolution in the two compartments . Prevalence of V3 mutations in X4/DM concordant DNA/RNA samples (N=8). All mutations found at the 35 V3 positions with an overall prevalence ≥ 10% were evaluated.
Conclus usions ns GTT provides highly concordant tropism prediction between pvDNA and plasma RNA , confirming that prediction of viral tropism using PBMCs DNA is accurate and feasible . Among discordant samples , the presence of X4/DM tropic viruses in pvDNA in the setting of low viremia levels suggests that the determination of genotypic tropism in pvDNA provides useful information for the optimization of the therapy in patients with low or suppressed viremia . ???????????? Further studies are needed to determine whether the use of MVC, in those patients suppressed and with discordant tropism in the two compartments (R5 in plasma RNA and X4/dual-mixed in pvDNA), could be effective over time. ????????????
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